CN105566510B - A kind of Portugal's oligosaccharides and its preparation method and application - Google Patents
A kind of Portugal's oligosaccharides and its preparation method and application Download PDFInfo
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Abstract
The invention discloses a kind of Portugal's oligosaccharides with butanedioic acid and pyruvoyl modification group and its modificationization derivative is removed, the main chain of Portugal's oligosaccharides is 8 glycosyls, unbranched connection, and the connection between main chain glycosyl includes a glycosidic bond of α 1,3 and 6 glycosidic bonds of β 1,3.After Portugal's oligosaccharides is by Liquid mixed fermentation agrobacterium ZX09 and series bacillus S09, separates and obtain directly from fermented liquid supernatant, fermented supernatant fluid centrifugation, after drying, obtains Portugal's oligosaccharides dry powder product by ethanol precipitation.The preparation method is fast and effective, technique is simple, and yield is high, and cost is cheap, pollution-free, suitable for industrialized production.Portugal's oligosaccharides good water solubility, molecular weight and structure prepared by the present invention are more homogeneous, easily prepared and purifying and bioactivity are high; can effective resistance of the activated plant to disease fungus; plant induced resistance agent and plant protection product can be used as; laminari-oligo saccharide is significantly better than to the Induced resistant effect of crop, is with a wide range of applications in the prevention of plant disease and control aspect.
Description
Technical field
The invention belongs to agriculture applied technology field, and in particular to Portugal's oligosaccharides made from a kind of microorganism mixed fermentation and its
Derivative, and the preparation method of Portugal's oligosaccharides and the application in terms of plant inducer is anti-with plant protection.
Background technology
Oligosaccharides (Oligosaccharide) is also known as oligosaccharide or oligosaccharide, usually passes through glycosidic bond by 3~10 monose
The glucide with straight or branched being formed by connecting.By body metabolism and it can absorb according to it and its have abiology work(
Can, oligosaccharides can be divided into common oligosaccharides and functional oligosaccharide.Common oligosaccharides such as sucrose, maltose, lactose and trehalose etc. can be by people
Body is digested and assimilated, and is energy matter, without special physiologic function.Functional oligosaccharide typically has low heat value, sweet, viscosity
Greatly, hygroscopicity it is strong, not by physicochemical properties such as pipe intestinal digestings, it is and soluble in water, have no toxic side effect, no antigen, body accumulation
Effect is weaker, has different physiological roles, for example improves intestinal microflora, improve food utilization efficiency, reduce serum courage
Sterol and neutral fat content, to improve immunity of organisms, antiviral, anti-inflammatory and plant inducer anti-etc..Wherein β-Portugal's oligosaccharide kind excites
The defense mechanism of sub- energy activated plant itself, induces the expression of plant disease-resistant signal transduction and related disease-resistant gene, and is referred to as
" plant immune vaccine ", there are great development and application potentiality.
The scale application of oligosaccharides still has difficulty at present, and its bottleneck factor essentially consists in source, the preparation side of oligosaccharides
Method and cost etc..The preparation method of oligosaccharides includes chemical synthesis, enzymatic synthesis and polysaccharide edman degradation Edman.Such as Chinese patent CN1373134A
β -1,3- Portugals the oligosaccharides that (β -1,3- glucooligosans and application) is obtained by chemical synthesis can be used as the bacteria remover of crops
For field of biological pesticide, but this method is related to the steps such as hydroxyl protection, deprotection and the chromatographic separation and purification of multistep, synthesis week
Phase is grown, and difficulty is larger, and yield is relatively low.At present macromolecular polysaccharide degraded approach mainly have chemical sour water solution, microorganism enzymolysis,
Thermal degradation, mechanical degradation, radiation degradation etc., most commonly sour water solution and enzyme hydrolysis method.Such as Chinese patent CN 102312021
The A preparation method of Derain oligosaccharides (a kind of can) employs prepared by acid hydrolyzation can Derain oligosaccharides.The product degree of polymerization that acid hydrolyzation obtains
Heterogeneity, molecular weight distribution are wider, it is difficult to isolate and purify, and yield is relatively low.Enzymatic isolation method generally has a selectivity, product structure compared with
To be homogeneous, glycosidic structure is not destroyed, and production technology cleans, accessory substance is few, free from environmental pollution, is the preferable way for preparing oligosaccharides
Footpath.But existing enzymatic isolation method is limited by enzyme source and species, the generally existing enzymolysis cycle is long, efficiency is low and production cost is high
The shortcomings of.
The content of the invention
It is an object of the invention to provide a kind of Portugal's oligosaccharides and its modificationization derivative is removed, and the preparation of these compounds
Method.The present invention above-mentioned Portugal's oligosaccharides is also provided and its go modificationization derivative plant inducer resist and protection in terms of in application.
To achieve these goals, technical scheme is as follows：
A kind of Portugal's oligosaccharides, its structural formula is shown in formula I：
Above-mentioned Portugal's oligosaccharides is to be formed by connecting by glucose unit by 6 β -1,3- glycosidic bonds and 1 α -1,3- glycosidic bond
Linear molecule, do not contain side chain, carry a succinyl modification group and a pyruvic acid modification group.
A kind of above-mentioned Portugal's oligosaccharides removes modificationization derivative by macromolecule alkali for hydrolysis removal succinyl modification group, its structure
Formula is as shown in Formula II：
A kind of method for removing modificationization derivative for preparing Portugal's oligosaccharides shown in above-mentioned Formula II, passes through macromolecule alkali for hydrolysis, institute
Alkali is NaOH or KOH.
What a kind of above-mentioned Portugal's oligosaccharides removed succinyl and pyruvic acid modification group by acid hydrolytic reaction simultaneously goes modificationization
Derivative, its structural formula is as shown in formula III：
A kind of method for removing modificationization derivative for preparing Portugal's oligosaccharides shown in above-mentioned formula III, passes through acid hydrolytic reaction, institute
Acid is hydrochloric acid, sulfuric acid, trifluoroacetic acid or acetic acid.
The present invention also provides the preparation method of above-mentioned Portugal's oligosaccharides, comprises the following steps：First by agrobacterium ZX09 and class
Respectively through solid medium rejuvenation, picking single bacterium drops down onto culture in seed liquor and obtains seed culture fluid bacillus S09, then will
The seed liquor of ZX09 and S09 bacterium is seeded in mixed fermentive culture medium successively, the shaken cultivation at 28~37 DEG C, and centrifugation afterwards is received
After collecting supernatant concentration, add ethanol precipitation and obtain Portugal's oligosaccharides crude product, Portugal's oligosaccharides crude product is obtained after Portugal's oligosaccharides crude product is lyophilized,
Refined Portugal's oligosaccharides is obtained finally by Gel filtration.
Heretofore described agrobacterium ZX09 is on January 21st, 2010 in China typical culture collection center
(CCTCC) preservation, deposit number are CCTCC NO：M2010020.Heretofore described series bacillus S09 is in 2012
It was preserved in China typical culture collection center (CCTCC) on May 30, preservation registration number is CCTCC M2012196.
Using the growth of sucrose substrate and synthesis of oligose precursor, two preferentially are carried out for series bacillus S09 by agrobacterium ZX09
Secondary metabolism and processing.Described agrobacterium ZX09 and series bacillus S09 seed liquor volume ratio are 1~5:1, agrobacterium
ZX09 prior to series bacillus S09 be seeded to mixed fermentive culture medium inoculation time it is poor be 0~36h.
The composition of described mixed fermentive culture medium is：20~40g/L of sucrose, 0.7~1.0g/L of urea, potassium dihydrogen phosphate
0.5~1.0g/L, 0.1~0.3g/L of anhydrous calcium chloride, 0.1~0.3g/L of bitter salt, green vitriol 0.01
~0.03g/L, 0.002~0.005g/L of zinc chloride, 0.001~0.003g/L of manganese sulfate, pH value are 5~10.
The time of described shaken cultivation is 48~72h.
Further, resist and answering in plant protection in plant inducer the invention provides above-mentioned Portugal widow carbohydrates and their derivative
With.Above-mentioned Portugal widow carbohydrates and their derivative can induce plant and produce disease-resistant factor, and promote plant growth, suppress disease, can
Resist and plant protection class product for preparing plant inducer.
A kind of above-mentioned Portugal widow carbohydrates and their derivative is in the anti-application with plant protection of plant inducer, its specific application process
For：It is 0.2~1.0g/L Portugal's oligosaccharides or derivatives thereof the aqueous solution by concentration, is uniformly sprayed on plant leaf blade surface.
Preferably, the concentration of described Portugal's oligosaccharides or derivatives thereof aqueous solution is 0.5g/L.
Portugal's oligosaccharides of the present invention is a kind of natural sugar chain molecule, reaches 85- for white or micro-yellow powder, sugared content
95%, protein content 3%-12%, water is highly soluble in, is had no toxic side effect, there is biodegradability, it is free from environmental pollution.It is above-mentioned
The preparation technology of Portugal's widow's carbohydrates and their derivative is simple, and cost is cheap, and yield is high, and product easily isolates and purifies, big beneficial to industrialization
Large-scale production.In addition, Portugal's oligosaccharides that the method obtains has no toxic side effect, bioactivity is higher, can effectively induce plant to produce disease-resistant
The factor, strengthen plant disease-resistant ability, compared with the laminari-oligo saccharide of same dose, oligosaccharides of the invention can more be effectively reduced soil
The incidence of disease of the crops such as beans, cucumber, Induced resistant effect are more notable.
Brief description of the drawings
Fig. 1 is the Portugal oligosaccharides I of present invention mass spectral analysis spectrogram.
Fig. 2 is the Portugal oligosaccharides I of the present invention two-dimentional nmr analysis spectrogram.
The Portugal oligosaccharides I that Fig. 3 is the present invention mitigates the effect contrast figure that Botrytis cinerea infects Arabidopsis leaf.
Fig. 4 is the intracellular H of Arabidopsis leaf after the Portugal oligosaccharides I processing of the present invention2O2With the horizontal variation diagram of salicylic acid.
Fig. 5 is intracellular pathogenesis-related proteins (PR1, resistance indicator protein after the Portugal oligosaccharides I of the present invention handles 36h；PR2,
Dextranase；PR3, chitinase；PR5, class thaumatin T) and Pdf1.2 (methyl jasmonate approach resistance protein) genetic transcription water
Flat variation diagram.
Fig. 6 is intracellular dextranase and the horizontal variation diagram of chitinase specific enzyme activity after the Portugal oligosaccharides I of the present invention handles 48h.
Embodiment
The present invention is described in further detail with reference to embodiment and accompanying drawing.
Embodiment 1
This example demonstrates that agrobacterium ZX09 and series bacillus S09 is carried out into Hybrid NC machine tool fermentation obtains Portugal's oligosaccharides
Method.
A. the preparation of seed liquor：ZX09 bacterium single bacterium colonies are seeded in inorganic salts-liquid sucrose culture medium, in 30 DEG C of vibrations
24h is cultivated, culture medium composition is：Sucrose 20g, sodium nitrate 0.5g, potassium dihydrogen phosphate 0.5g, anhydrous calcium chloride 0.1g, seven hydrations
Magnesium sulfate 0.3g, green vitriol 0.02g, zinc chloride 0.003g, manganese sulfate 0.001g, water 1000mL, pH 7.0.121
DEG C autoclaving 20min.S09 bacterium single bacterium colonies are seeded in LBO fluid nutrient mediums, in 35 DEG C of shaken cultivation 24h, culture medium group
As sodium chloride 0.5g, yeast extract 0.5g, peptone 1.0g, oatmeal 0.5g, 121 DEG C of high pressure steam sterilization 20min.
B. Hybrid NC machine tool：The seed liquor of cultured ZX09 bacterium and S09 bacterium is pressed 3:1 ratio is seeded to mixed fermentation training
Support in base, in 30 DEG C of shaken cultivation 48h, the composition of mixed fermentive culture medium is：Sucrose 20g, urea 0.8g, potassium dihydrogen phosphate
0.75g, anhydrous calcium chloride 0.15g, bitter salt 0.15g, green vitriol 0.02g, zinc chloride 0.003g, sulfuric acid
Manganese 0.0015g, water 1000mL, pH 7.0.121 DEG C of high pressure steam sterilization 20min.
C. the separation and purifying of Portugal's oligosaccharides：Mixed fungus fermentation supernatant is collected, after being concentrated by Rotary Evaporators, using ethanol
Portugal's oligosaccharide compositions in zymotic fluid are precipitated, Portugal's oligosaccharides crude product is obtained after freezing.Chong Rong Portugals oligosaccharides crude product, it is molten to be made into 20% Portugal's oligosaccharides
After liquid, Sephadex G25 gel chromatography columns are crossed, remove the impurity such as high molecular weight protein and small molecule sucrose, are monitored using DNS methods
Reduced sugar goes out peak position, collects eluent, after freezing, obtains purifying Portugal's oligosaccharides dry powder.
It is utilized respectively the information such as molecular weight and the structure of mass spectrum and nuclear magnetic resonance parsing Portugal's oligosaccharides.Show from mass spectrometry results
The molecular weight for showing Portugal's oligosaccharides is (741*2+2)=1484Da, with two negative electrical charges, shows the presence of two modification groups
(Fig. 1).By two-dimensional nucleus magnetic spectrum elucidation, the further backbone structure, modification group and its position (Fig. 2, a for obtaining Portugal's oligosaccharides
It is1H-1H chemical shift correlations COSY is composed, and b is heteronuclear Multiple-quantum correlation HMQC spectrums, and c is heteronuclear list quantum correlation hsqc spectrum).
Embodiment 2
The preparation of Portugal's oligosaccharide derivative.
Portugal's oligosaccharides after purification is configured to the 10mg/mL aqueous solution, adds final concentration of 0.2M NaOH reaction 2-5h,
After being neutralized with HCl, cross Sephadex G25 and carry out desalination, collect Portugal oligosaccharides eluted peak sample, obtain and remove succinyl modification base
The Portugal oligosaccharide derivative II of group.Portugal's oligosaccharides after purification is configured to the 10mg/mL aqueous solution, adds final concentration of 0.5M's
HCl reacts 5h, after being neutralized with NaOH, crosses Sephadex G25 and carries out desalination, collect Portugal oligosaccharides eluted peak sample, acquisition is gone simultaneously
The Portugal oligosaccharide derivative III modified except succinyl and pyruvic acid.
Embodiment 3
Portugal's oligosaccharides infects the control of arabidopsis to Botrytis cinerea.
0.2-1.0g/L Portugal's oligosaccharides aqueous solution is prepared, is uniformly sprayed to the arabidopsis thaliana blade for growing into suitable size
On, spray once within one day, to spray clear water as control.Portugal's oligosaccharides is pre-processed two days later, by the Botrytis cinerea of culture solid
Punched on body culture medium, mold agar block is labelled in Arabidopsis leaf and carries out infecting culture.Control group and Portugal are observed after 1-3 days
The gradient of infection of oligosaccharides group plant leaf blade.
As a result show, arabidopsis thaliana leaves infected rate can be reduced by spraying Portugal's oligosaccharide solution in advance, and germ patch is obvious
Diminish.Arabidopsis thaliana blade figure after Portugal's oligosaccharide solution that Fig. 3 show sprinkling clear water and spray concentration is 0.5g/L in advance.
Embodiment 4
Induction of Portugal's oligosaccharides to arabidopsis resistance.
0.2-1.0g/L Portugal's oligosaccharides aqueous solution is prepared, is uniformly sprayed to the arabidopsis thaliana blade for growing into suitable size
On, after different time, clip blade is immediately placed in liquid nitrogen after weighing and frozen.
The horizontal measure of blade cell hydrogen peroxide：Weigh 100mg blades, add acetone high-speed homogenization, in centrifuging and taking
Clear 200 μ L, add 600 μ L dipotassium hydrogen phosphates buffer solutions (50mM, pH 8.2), and vibration mixes.Acutely shaken after adding 1mL chloroforms
Afterwards, the μ L upper strata aqueous phases of centrifuging and taking 500, add tiron and mix, add the vibration of 200 μ L concentrated ammonia liquors, precipitation is collected by centrifugation.With
After 500 μ L 2M sulfuric acid solutions dissolving precipitation, absorbance is determined under 415nm wavelength.
The horizontal measure of salicylic acid (SA) in blade cell：Blade 100mg is taken, adds centrifuging and taking after the μ L of methanol 500 homogenate
Supernatant, dried up with nitrogen evaporator；Add 300 μ L5% trichloroacetic acids dissolving precipitation；Add 550 μ L Ethyl acetate-cyclohexanes (1：1)
Solution extracts, and centrifuges aqueous phase and organic phase, transfer upper organic phase is into clean centrifuge tube.After repeatedly this is operated 2-3 times
Merge upper layer of extraction liquid, methanol is dissolved after being dried up with nitrogen evaporator, and free state SA contents are detected with HPLC.Added in lower floor's aqueous phase
200 μ L 8M hydrochloric acid solutions, 80 DEG C of water-bath 1h make reference state SA be changed into free state SA, and reference state SA contents are detected with HPLC.
The measure of intracellular PR gene transcriptional level：100mg blades are weighed, leaf is extracted with Trizol kits
Piece total serum IgE, with Invitrogen Reverse Transcriptase kit by RNA reverse transcriptions into after cDNA, using poly ubiquitin enzyme UBQ5 genes as
Internal reference, real-time fluorescence quantitative PCR reaction (QRT-PCR) is carried out, the expression of intracellular pathogenesis-related proteins is analyzed from transcriptional level
Situation.
Intracellular dextranase and chitinase activity measure：Appropriate blade is ground in liquid nitrogen it is broken after, with containing albumen
Phosphate buffer (pH 6.5, the 0.1M) dissolving of Protease Inhibitor Cocktail mixes 1h；It is crude enzyme liquid that supernatant, which is collected by centrifugation,.Point
1h is not incubated at 37 DEG C as substrate using 1% laminarin and 1% chitin solution and carries out enzyme reaction.Determined with DNS methods
The growing amount of reduced sugar in reaction solution.One enzyme-activity unit is defined as in 1h from substrate required for 1 μm of ol glucose of release
Enzyme amount.
As a result show, after Portugal's oligosaccharide solution processing arabidopsis, H in blade cell can be significantly improved2O2It is horizontal with salicylic acid,
Strengthen PR gene and Pdf1.2 transcriptional level, and the synthesis of intracellular dextranase and chitinase can be promoted.
Result figure after Portugal's oligosaccharide solution processing arabidopsis that it is 0.5g/L with concentration that Fig. 4, Fig. 5 and Fig. 6, which are,.It is 0.5g/L's with concentration
After Portugal's oligosaccharide solution processing arabidopsis, H in blade cell can be significantly improved2O2It is horizontal (Fig. 4) with salicylic acid；It is related to strengthen the course of disease
GFP (PR1, resistance indicator protein gene；PR2, glucanase gene；PR3, chitinase gene；PR5, class thaumatin T
Gene) and Pdf1.2 (methyl jasmonate approach resistance protein gene) transcriptional level (Fig. 5), and intracellular dextranase can be promoted
With the synthesis (Fig. 6) of chitinase.To sum up result illustrates, microorganism Portugal oligosaccharides of the invention can fast and effectively excite plant
A series of interior metabolic regulation systems, disease resistance of plant is induced, strengthen plant disease-resistant and anti-adversity ability, had and be used as plant immune
The application potential of inducer and plant protection agent.
Embodiment 5
The field test that Portugal's widow's carbohydrates and their derivative prevention crop disease occurs.
Portugal's oligosaccharides or derivatives thereof is configured to 10% mother liquor, by 200 times of dilutions during use.To different crop (soil
Beans, cucumber, tobacco, tomato) carry out spray test.Using clear water processing as negative control, with the enzymolysis product of laminarin
Laminari-oligo saccharide is positive control, and daily sprinkling once, is preferred with blade wetting No drip type.Severity Scaling standard is：It is 0 grade, disease-free
Spot；1 grade, lesion area accounts for whole leaf area below 5%；3 grades, lesion area accounts for whole leaf area 6%~10%；5 grades, scab
Area accounts for whole leaf area 11%~20%；7 grades, lesion area accounts for whole leaf area 21%~50%；9 grades, lesion area accounts for
Whole leaf area more than 50%.
The incidence of disease=morbidity strain number/investigation strain number × 100%
Disease index=Σ (the sick numbers of sheets at different levels × relative value of series)/(investigation total number of sheets × highest typical value) × 100
Disease resisting effect=(control disease index-processing disease index)/control disease index × 100%
Portugal's widow's carbohydrates and their derivative is shown in Table 1 to the Induced resistant effect of Different Crop, and Portugal oligosaccharides I is with laminari-oligo saccharide to Different Crop
Induced resistant effect be shown in Table 2.
The Portugal's widow's carbohydrates and their derivative of table 1 compares the Induced resistant effect of Different Crop
From table 1 it follows that this several crop applying oligosaccharides or derivatives thereof to potato, cucumber, tobacco and tomato
Afterwards, the incidence of disease and disease index are decreased obviously, and disease resistance substantially increases.Wherein, oligosaccharides I Induced resistant effect outline is gone higher than it
Modificationization derivative I I and III.Using during oligosaccharides, no adverse side effect, occur without bad poisoning phenomenon.
The Portugal oligosaccharides I of table 2 is compared with laminari-oligo saccharide is to the Induced resistant effect of Different Crop
From Table 2, it can be seen that Portugal oligosaccharides I and laminari-oligo saccharide can reduce potato, cucumber, tobacco and tomato, this is several
The incidence of disease and disease index of crop.Under same dose, Portugal oligosaccharides I Induced resistant effect will be substantially better than laminari-oligo saccharide.With sea
Band oligosaccharides is compared, and Portugal oligosaccharides preparation technology of the invention is simple, and production cost is low, and product structure is homogeneous, and anti-effect is lured to crop
Fruit is more notable, has the application potential as plant immune inducer and plant protection agent.
Claims (10)
1. a kind of Portugal's oligosaccharides, its structural formula is shown in formula I：
Portugal's oligosaccharides is the line being formed by connecting by glucose unit by 6 β -1,3- glycosidic bonds and 1 α -1,3- glycosidic bond
Property molecule, do not contain side chain, carry a succinyl modification group and a pyruvic acid modification group.
2. the derivative of Portugal's oligosaccharides as claimed in claim 1, its structural formula is as shown in Formula II or formula III：
3. the preparation method of the derivative of Portugal's oligosaccharides as claimed in claim 2, it is characterised in that obtained by macromolecule alkali for hydrolysis
The derivative of Portugal's oligosaccharides shown in Formula II, alkali used are NaOH or KOH；The Portugal shown in formula III is obtained by acid hydrolytic reaction
The derivative of oligosaccharides, acid used are hydrochloric acid, sulfuric acid, trifluoroacetic acid or acetic acid.
4. the preparation method of Portugal's oligosaccharides as claimed in claim 1, it is characterised in that comprise the following steps：First by soil bar
Respectively through solid medium rejuvenation, picking single bacterium drops down onto culture in seed culture medium and planted bacterium ZX09 and series bacillus S09
Sub- nutrient solution, then the seed culture fluid of ZX09 and S09 bacterium is seeded in mixed fermentive culture medium successively, at 28~37 DEG C
Shaken cultivation, after supernatant concentration is collected by centrifugation afterwards, adds ethanol precipitation and obtain Portugal's oligosaccharides crude product, Portugal's oligosaccharides crude product freezes
Portugal's oligosaccharides crude product is obtained after dry, refined Portugal's oligosaccharides is obtained finally by gel permeation chromatography.
5. the preparation method of Portugal's oligosaccharides as claimed in claim 4, it is characterised in that described agrobacterium ZX09 and class gemma
Bacillus S09 seed culture fluid volume ratio is 1~5:1, agrobacterium ZX09 is seeded to mixed fermentation prior to series bacillus S09
The inoculation time difference of culture medium is 0~36h.
6. the preparation method of Portugal's oligosaccharides as claimed in claim 4, it is characterised in that the composition of described mixed fermentive culture medium
For：20~40g/L of sucrose, 0.7~1.0g/L of urea, 0.5~1.0g/L of potassium dihydrogen phosphate, 0.1~0.3g/L of anhydrous calcium chloride,
0.1~0.3g/L of bitter salt, 0.01~0.03g/L of green vitriol, 0.002~0.005g/L of zinc chloride, sulphur
Sour 0.001~0.003g/L of manganese, pH value are 5~10.
7. the preparation method of Portugal's oligosaccharides as claimed in claim 4, it is characterised in that the time of described shaken cultivation be 48~
72h。
8. the derivative of Portugal's oligosaccharides described in Portugal's oligosaccharides as claimed in claim 1 or claim 2 as plant induced resistance agent and
Plant protection product is in the anti-application with plant protection of plant inducer.
9. Portugal's oligosaccharides as claimed in claim 8 or derivatives thereof as plant induced resistance agent and plant protection product plant inducer it is anti-and
Application in plant protection, it is characterised in that specifically application process is：By concentration be 0.2~1.0g/L Portugal's oligosaccharides or its
The aqueous solution of derivative, uniformly it is sprayed on plant leaf blade surface.
10. Portugal's oligosaccharides as claimed in claim 9 or derivatives thereof resists as plant induced resistance agent and plant protection product in plant inducer
With the application in plant protection, it is characterised in that the concentration of the aqueous solution of described Portugal's oligosaccharides or derivatives thereof is 0.5g/L.
Priority Applications (1)
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